Methods and kits for the detection of viral infections

Abstract

The invention relates to an improved assay for detecting antibodies in a tissue sample from individuals who test seronegative by conventional assay techniques, thus aiding in the diagnosis of possible pathogenic infections. Specifically, the invention relates to improved assay methods and kits that enable efficient detection of antibodies against a viral infection.

What is claimed is: 
     
         1 . A formulation comprising a medium for incubating a whole blood sample in a culture, wherein said medium comprises (a) one or more activators of (i) virus-primed lymphocytes, (ii) memory cells specific for said virus, (iii) virus-specific antibody production, or (iv) a combination thereof, and (b) a non-carbon dioxide-dependent buffering agent. 
     
     
         2 . The formulation of  claim 1 , wherein said buffering agent is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). 
     
     
         3 . The formulation of  claim 1 , wherein said buffering agent is a phosphate-based buffer. 
     
     
         4 . The formulation of  claim 3 , wherein said phosphate-based buffer is L15 Medium. 
     
     
         5 . The formulation of any one of the preceding claims, wherein said activator is pokeweed mitogen. 
     
     
         6 . The formulation of any one of the preceding claims, wherein said activator is a viral-derived peptide, lectin, bacterial endotoxin, a virus, lipid A, a cytokine, a lymphokine, or a combination thereof. 
     
     
         7 . A container apparatus comprising a medium for incubating a whole blood sample in a culture, wherein said medium comprises (a) one or more activators of (i) virus-primed lymphocytes, (ii) memory cells specific for said virus, (iii) virus-specific antibody production, or (iv) a combination thereof and (b) a non-carbon dioxide-dependent buffering agent. 
     
     
         8 . The container of  claim 7 , wherein said buffering agent is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). 
     
     
         9 . The container of  claim 7 , wherein said buffering agent is a phosphate-based buffer. 
     
     
         10 . The container of  claim 9 , wherein said phosphate-based buffer is L15 Medium. 
     
     
         11 . The container of any one of  claims 7 - 10 , wherein said activator is pokeweed mitogen. 
     
     
         12 . The container of any one of  claims 7 - 10 , wherein said activator is a viral-derived peptide, lectin, bacterial endotoxin, a virus, lipid A, a cytokine, or a lymphokine. 
     
     
         13 . The container of any one of  claims 7 - 12 , wherein said container comprises a polymer that is non-toxic to cells in said culture. 
     
     
         14 . The container of  claim 13 , wherein said polymer is a polystyrene polymer. 
     
     
         15 . The container of  claim 13 , wherein said polymer is a polypropylene polymer. 
     
     
         16 . The container of any one of  claims 7 - 15 , wherein said container is capable of stably maintaining liquid and/or vacuum pressure inside said container. 
     
     
         17 . The container of any one of  claims 7 - 16 , wherein said container has a cap that has a plurality of positioning mechanisms. 
     
     
         18 . The container of  claim 17 , wherein said container has a first and second positioning mechanisms, wherein said first positioning mechanism renders the container fully closed, thereby stably maintaining liquid and/or vacuum pressure inside said container and wherein said second positioning mechanism provides ventilation to the contents of said container while preserving a sterile environment inside said container. 
     
     
         19 . The container of any one of  claims 7 - 18 , wherein said container is made of a plastic, glass, silicon, synthetic membrane, or metal. 
     
     
         20 . The container of any one of  claims 7 - 19 , wherein said container comprises a treatment and/or coating of the inner surface. 
     
     
         21 . The container of any one of  claims 7 - 20 , wherein said container is a test tube, a bottle, a well or a flask. 
     
     
         22 . The container of any one of  claims 7 - 21 , wherein said container is vacuum-sealed. 
     
     
         23 . The container of  claim 22 , wherein said container is a vacutube. 
     
     
         24 . A method for detecting virus-specific antibodies in a sample from a subject in the absence of CO 2  enrichment, the method comprising:
 a) incubating a whole blood sample from said subject in a culture in the presence of a medium, said medium comprising one or more activators of (i) lymphocytes primed by said virus, (ii) memory cells specific for said virus, (iii) antibody production against said virus, or (iv) a combination thereof, and a non-carbon dioxide-dependent buffering agent;   b) exposing the resultant culture of step a) to a viral antigen, thereby allowing an antigen-antibody immune complex to form; and   c) detecting the antigen-antibody immune complex of step b);   thereby detecting the presence of viral-specific antibodies following a culture step in the absence of CO2 enrichment.   
     
     
         25 . The method of  claim 24 , wherein said incubating step is performed in the absence of a CO 2  incubator. 
     
     
         26 . The method of any one of  claims 24 - 25 , wherein said buffering agent is HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). 
     
     
         27 . The method of any one of  claims 24 - 26  wherein said buffering agent is a phosphate-based buffering agent. 
     
     
         28 . The method of  claim 27 , wherein said phosphate-based buffer is L15 Medium. 
     
     
         29 . The method of any one of  claims 24 - 28  wherein the culture of step a) results in a supernatant, and the supernatant is exposed to a viral antigen in step b), thereby allowing an antigen-antibody immune complex to form. 
     
     
         30 . The method of any one of  claims 24 - 29  wherein said virus is a retrovirus. 
     
     
         31 . The method of  claim 30 , wherein said retrovirus is human immunodeficiency virus (HIV). 
     
     
         32 . The method of any one of  claims 24 - 29  wherein said virus is a xenotropic murine leukemia virus (XMRV). 
     
     
         33 . The method of any one of  claims 24 - 29  wherein said virus is a hepatitis C virus (HCV). 
     
     
         34 . The method of any one of  claims 24 - 29  wherein said virus is a hepatitis B virus (HBV). 
     
     
         35 . The method of any one of  claims 24 - 29  wherein said virus is a hepatitis A virus (HAV), hepatitis D virus (HDV), or hepatitis E virus (HEV). 
     
     
         36 . The method of any one of  claims 24 - 29  wherein the culture of step a) further comprises an antibody against a B-lymphocyte membrane domain. 
     
     
         37 . The method of  claim 36 , wherein said antibody is anti-IgD. 
     
     
         38 . The method of  claim 36 , wherein said antibody is anti-IgG, anti-IgA or anti-IgE. 
     
     
         39 . The method of  claim 36 , wherein said antibody is anti-CD19, anti-CD10, anti-CD23, anti-CD25, or anti-CD40. 
     
     
         40 . The method of any one of  claims 24 - 39 , wherein said activator is pokeweed mitogen. 
     
     
         41 . The method of any one of  claims 24 - 39 , wherein said activator is a viral-derived peptide, lectin, bacterial endotoxin, a virus, lipid A, a cytokine, or a lymphokine. 
     
     
         42 . The method of any one of  claims 24 - 41 , wherein said virus-primed lymphocytes or memory cells are B-lymphocytes. 
     
     
         43 . The method of any one of  claims 24 - 42 , wherein said virus-primed lymphocytes or memory cells are T-lymphocytes. 
     
     
         44 . The method of any one of  claims 24 - 43 , further comprising the step of collecting whole blood sample from said subject, prior to step a). 
     
     
         45 . The method of  claim 44 , wherein said whole blood sample is collected into a container. 
     
     
         46 . The method of  claim 45 , wherein said container comprises said medium. 
     
     
         47 . The method of any one of  claims 45 - 46 , wherein said container comprises a polymer that is non-toxic to cells in said culture. 
     
     
         48 . The method of  claim 47 , wherein said polymer is a polystyrene polymer. 
     
     
         49 . The method of  claim 47 , wherein said polymer is a polypropylene polymer. 
     
     
         50 . The method of any one of  claims 45 - 49 , wherein said container is capable of stably maintaining liquid and/or vacuum pressure inside said container. 
     
     
         51 . The method of any one of  claims 45 - 50 , wherein said container has a cap that has a plurality of positioning mechanisms. 
     
     
         52 . The method of  claim 51 , wherein said container has a first and second positioning mechanisms, wherein said first positioning mechanism renders the container fully closed, thereby stably maintaining liquid and/or vacuum pressure inside said container and wherein said second positioning mechanism provides ventilation to the contents of said container while preserving a sterile environment inside said container. 
     
     
         53 . The method of any one of  claims 24 - 52 , wherein the method further comprises the steps of: (i) collecting a first blood sample prior to the incubation step; (ii) measuring the level of antibodies in said first blood sample; (iii) collecting a second blood sample after the incubation; (iv) measuring the level of antibodies in said second blood sample; and (v) comparing the measurements of the levels of antibodies between said first and second blood samples. 
     
     
         54 . The method of any one of  claims 24 - 52 , wherein the method further comprises the steps of: (i) collecting a first blood sample prior to the incubation step and storing said blood assay sample; (ii) collecting a second blood sample after said incubation step; (iii) measuring the level of antibodies in said first and said second blood samples concurrently; and (iv) comparing the measurements of the levels of antibodies between said first and second blood samples. 
     
     
         55 . The method of any one of  claims 24 - 54 , wherein said antigen is added to said culture to shorten the incubation time and/or to provide diagnosis in situ. 
     
     
         56 . The method of any one of  claims 24 - 55 , wherein said antigen-antibody immune complex is detected on a solid support. 
     
     
         57 . The method of  claim 56 , wherein said solid support is a nitrocellulose strip. 
     
     
         58 . The method of any one of  claims 24 - 57 , wherein said subject has a suspected viral infection. 
     
     
         59 . A method for diagnosing a viral infection caused by a virus in a subject, the method comprising:
 a) collecting a whole blood sample from said subject;   b) incubating said blood sample, in the absence of CO 2  enrichment, in a culture in the presence of a medium, said medium comprising one or more activators of (i) lymphocytes primed by said virus, (ii) memory cells specific for said virus, (iii) antibody production against said virus, or (iv) a combination thereof, and a non-carbon dioxide-dependent buffering agent;   c) exposing the resultant culture of step b) to an antigen of said virus, thereby allowing an antigen-antibody immune complex to form; and   d) detecting the antigen-antibody immune complex of step c);   wherein the detection of said antigen-antibody immune complex indicates said subject has been infected with said virus.   
     
     
         60 . The method of  claim 59 , wherein said viral infection is a chronic viral infection, a recent viral infection, a very early (WP) viral infection, a latent viral infection, a viral re-infection, a viral breakthrough infection, or a combination thereof. 
     
     
         61 . The method of any one of  claims 59 - 60 , wherein said incubating step is performed in the absence of a CO 2  incubator. 
     
     
         62 . The method of any one of  claims 59 - 61 , wherein said buffering agent is HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). 
     
     
         63 . The method of any one of  claims 59 - 61 , wherein said buffering agent is a phosphate-based buffering agent. 
     
     
         64 . The method of  claim 63 , wherein said phosphate-based buffer is L15 Medium. 
     
     
         65 . The method of any one of  claims 59 - 64 , wherein the culture of step b) results in a supernatant, and the supernatant is exposed to a viral antigen in step c), thereby allowing an antigen-antibody immune complex to form. 
     
     
         66 . The method of any one of  claims 59 - 65 , wherein said virus is a retrovirus. 
     
     
         67 . The method of  claim 66 , wherein said retrovirus is human immunodeficiency virus (HIV). 
     
     
         68 . The method of any one of  claims 59 - 65 , wherein said virus is a xenotropic murine leukemia virus (XMRV). 
     
     
         69 . The method of any one of  claims 59 - 65 , wherein said virus is a hepatitis C virus (HCV). 
     
     
         70 . The method of any one of  claims 59 - 65 , wherein said virus is a hepatitis B virus (HBV). 
     
     
         71 . The method of any one of  claims 59 - 65 , wherein said virus is a hepatitis A virus (HAV), hepatitis D virus (HDV), or hepatitis E virus (HEV). 
     
     
         72 . The method of any one of  claims 59 - 71 , wherein the culture of step b) further comprises an antibody against a B-lymphocyte membrane domain. 
     
     
         73 . The method of  claim 72 , wherein said antibody is anti-IgD. 
     
     
         74 . The method of  claim 72 , wherein said antibody is anti-IgG, anti-IgA or anti-IgE. 
     
     
         75 . The method of  claim 72 , wherein said antibody is anti-CD19, anti-CD10, anti-CD23, anti-CD25, or anti-CD40. 
     
     
         76 . The method of any one of  claims 59 - 75 , wherein said activator is pokeweed mitogen. 
     
     
         77 . The method of any one of  claims 59 - 75 , wherein said activator is a viral-derived peptide, lectin, bacterial endotoxin, a virus, lipid A, a cytokine, or a lymphokine. 
     
     
         78 . The method of any one of  claims 59 - 77 , wherein said virus-primed lymphocytes or memory cells are B-lymphocytes. 
     
     
         79 . The method of any one of  claims 59 - 78 , wherein said virus-primed lymphocytes or memory cells are T-lymphocytes. 
     
     
         80 . The method of any one of  claims 59 - 79 , wherein said whole blood sample is collected into a container. 
     
     
         81 . The method of  claim 80 , wherein said container comprises said medium. 
     
     
         82 . The method of any one of  claims 80 - 81 , wherein said container comprises a polymer that is non-toxic to cells in said culture. 
     
     
         83 . The method of  claim 82 , wherein said polymer is a polystyrene polymer. 
     
     
         84 . The method of  claim 82 , wherein said polymer is a polypropylene polymer. 
     
     
         85 . The method of any one of  claims 80 - 84 , wherein said container is capable of stably maintaining liquid and/or vacuum pressure inside said container. 
     
     
         86 . The method of any one of  claims 80 - 85 , wherein said container has a cap that has a plurality of positioning mechanisms. 
     
     
         87 . The method of  claim 86 , wherein said container has a first and second positioning mechanisms, wherein said first positioning mechanism renders the container fully closed, thereby stably maintaining liquid and/or vacuum pressure inside said container and wherein said second positioning mechanism provides ventilation to the contents of said container while preserving a sterile environment inside said container. 
     
     
         88 . The method of any one of  claims 59 - 87 , wherein the method further comprises the steps of: (i) collecting a first blood sample prior to the incubation step; (ii) measuring the level of antibodies in said first blood sample; (iii) collecting a second blood sample after the incubation; (iv) measuring the level of antibodies in said second blood sample; and (v) comparing the measurements of the levels of antibodies between said first and second blood samples. 
     
     
         89 . The method of any one of  claims 59 - 87 , wherein the method further comprises the steps of: (i) collecting a first blood sample prior to the incubation step and storing said first blood sample; (ii) collecting a second blood sample after said incubation step; (iii) measuring the level of antibodies in said first and said second blood samples concurrently; and (iv) comparing the measurements of the levels of antibodies between said first and second blood samples. 
     
     
         90 . The method of any one of  claims 59 - 89 , wherein said antigen is added to said culture to shorten the incubation time and/or to provide diagnosis in situ. 
     
     
         91 . The method of any one of  claims 59 - 90 , wherein said antigen-antibody immune complex is detected on a solid base. 
     
     
         92 . The method of  claim 91 , wherein said solid base is a nitrocellulose strip. 
     
     
         93 . A method for increasing low virus-specific antibody levels in a whole blood sample from a subject to a detectable level comprising:
 a) collecting said whole blood sample from said subject; and   b) incubating said sample, in the absence of CO 2  enrichment, in a culture in the presence of a medium, said medium comprising one or more activators of (i) lymphocytes primed by said virus, (ii) memory cells specific for said virus, (iii) antibody production against said virus, or (iv) a combination thereof, and a non-carbon dioxide-dependent buffering agent;   thereby increasing low virus-specific antibody levels to a detectable level in said sample.   
     
     
         94 . The method of  claim 93 , wherein said incubating step is performed in the absence of a CO 2  incubator. 
     
     
         95 . The method of any one of  claims 93 - 94 , wherein said buffering agent is HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). 
     
     
         96 . The method of any one of  claims 93 - 94 , wherein said buffering agent is a phosphate-based buffering agent. 
     
     
         97 . The method of  claim 96 , wherein said phosphate-based buffer is L15 Medium. 
     
     
         98 . The method of any one of  claims 93 - 97 , wherein said virus is a retrovirus. 
     
     
         99 . The method of  claim 98 , wherein said retrovirus is human immunodeficiency virus (HIV). 
     
     
         100 . The method of any one of  claims 93 - 97 , wherein said virus is a xenotropic murine leukemia virus (XMRV). 
     
     
         101 . The method of any one of  claims 93 - 97 , wherein said virus is a hepatitis C virus (HCV). 
     
     
         102 . The method of any one of  claims 93 - 97 , wherein said virus is a hepatitis B virus (HBV). 
     
     
         103 . The method of any one of  claims 93 - 97 , wherein said virus is a hepatitis A virus (HAV), hepatitis D virus (HDV), or hepatitis E virus (HEV). 
     
     
         104 . The method of any one of  claims 93 - 103 , wherein the culture of step b) further comprises an antibody against a B-lymphocyte membrane domain. 
     
     
         105 . The method of  claim 104 , wherein said antibody is anti-IgD. 
     
     
         106 . The method of  claim 104 , wherein said antibody is anti-IgG, anti-IgA or anti-IgE. 
     
     
         107 . The method of  claim 104 , wherein said antibody is anti-CD19, anti-CD10, anti-CD23, anti-CD25, or anti-CD40. 
     
     
         108 . The method of any one of  claims 93 - 107 , wherein said activator is pokeweed mitogen. 
     
     
         109 . The method of any one of  claims 93 - 107 , wherein said activator is a viral-derived peptide, lectin, bacterial endotoxin, a virus, lipid A, a cytokine, or a lymphokine. 
     
     
         110 . The method of any one of  claims 93 - 109 , wherein said virus-primed lymphocytes or memory cells are B-lymphocytes. 
     
     
         111 . The method of any one of  claims 93 - 110 , wherein said virus-primed lymphocytes or memory cells are T-lymphocytes. 
     
     
         112 . The method of any one of  claims 93 - 111 , wherein said whole blood sample is collected into a container. 
     
     
         113 . The method of  claim 112 , wherein said container comprises said medium. 
     
     
         114 . The method of any one of  claims 112 - 113 , wherein said container comprises a polymer that is non-toxic to cells in said culture. 
     
     
         115 . The method of  claim 114 , wherein said polymer is a polystyrene polymer. 
     
     
         116 . The method of  claim 114 , wherein said polymer is a polypropylene polymer. 
     
     
         117 . The method of any one of  claims 112 - 116 , wherein said container is capable of stably maintaining liquid and/or vacuum pressure inside said container. 
     
     
         118 . The method of any one of  claims 112 - 117 , wherein said container has a cap that has a plurality of positioning mechanisms. 
     
     
         119 . The method of  claim 118 , wherein said container has a first and second positioning mechanisms, wherein said first positioning mechanism renders the container fully closed, thereby stably maintaining liquid and/or vacuum pressure inside said container and wherein said second positioning mechanism provides ventilation to the contents of said container while preserving a sterile environment inside said container. 
     
     
         120 . The method of any one of  claims 93 - 119 , wherein said subject has a suspected viral infection. 
     
     
         121 . A kit for the detection of virus-specific antibodies, the kit comprising: a container for retaining and culturing a whole blood sample, wherein said container comprises a mechanism for incubating said sample without any CO 2  enrichment, and wherein said container further comprises a medium, said medium comprising one or more activators of (i) lymphocytes primed by said virus, (ii) memory cells specific for said virus, (iii) antibody production against said virus, or (iv) a combination thereof, and a non-carbon dioxide-dependent buffering agent. 
     
     
         122 . The kit of  claim 121 , wherein said kit additionally comprises an assay for the detection of viral-specific antibodies. 
     
     
         123 . The kit of  claim 122 , wherein said assay is an enzyme linked immunosorbent assay, a western blot, a lateral flow, or an immunofluorescence assay. 
     
     
         124 . The kit of any one of  claims 121 - 123 , wherein said buffering agent is HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). 
     
     
         125 . The kit of any one of  claims 121 - 123 , wherein said buffering agent is a phosphate-based buffering agent. 
     
     
         126 . The kit of  claim 125 , wherein said phosphate-based buffer is L15 Medium. 
     
     
         127 . The kit of any one of  claims 121 - 126 , wherein said virus is a retrovirus. 
     
     
         128 . The kit of  claim 127 , wherein said retrovirus is human immunodeficiency virus (HIV). 
     
     
         129 . The kit of any one of  claims 121 - 126 , wherein said virus is a xenotropic murine leukemia virus (XMRV). 
     
     
         130 . The kit of any one of  claims 121 - 126 , wherein said virus is a hepatitis C virus (HCV). 
     
     
         131 . The kit of any one of  claims 121 - 126 , wherein said virus is a hepatitis B virus (HBV). 
     
     
         132 . The kit of any one of  claims 121 - 126 , wherein said virus is a hepatitis A virus (HAV), hepatitis D virus (HDV), or hepatitis E virus (HEV). 
     
     
         133 . The kit of any one of  claims 121 - 132 , wherein the medium further comprises an antibody against a B-lymphocyte membrane domain. 
     
     
         134 . The kit of  claim 133 , wherein said antibody is anti-IgD. 
     
     
         135 . The kit of  claim 133 , wherein said antibody is anti-IgG, anti-IgA or anti-IgE. 
     
     
         136 . The kit of  claim 133 , wherein said antibody is anti-CD19, anti-CD10, anti-CD23, anti-CD25, or anti-CD40. 
     
     
         137 . The kit of any one of  claims 121 - 136 , wherein said activator is pokeweed mitogen. 
     
     
         138 . The kit of any one of  claims 121 - 136 , wherein said activator is a viral-derived peptide, lectin, bacterial endotoxin, a virus, lipid A, a cytokine, or a lymphokine. 
     
     
         139 . The kit of any one of  claims 121 - 138 , wherein said virus-primed lymphocytes or memory cells are B-lymphocytes. 
     
     
         140 . The kit of any one of  claims 121 - 139 , wherein said virus-primed lymphocytes or memory cells are T-lymphocytes. 
     
     
         141 . The kit of any one of  claims 121 - 140 , wherein said container comprises a polymer non-toxic to cells in said culture. 
     
     
         142 . The kit of  claim 141 , wherein said polymer is a polystyrene polymer. 
     
     
         143 . The kit of  claim 141 , wherein said polymer is a polypropylene polymer. 
     
     
         144 . The kit of any one of  claims 121 - 143 , wherein said container is capable of stably maintaining liquid and/or vacuum pressure inside said container. 
     
     
         145 . The kit of any one of  claims 121 - 143 , wherein said container has a cap that has a plurality of positioning mechanisms. 
     
     
         146 . The kit of  claim 145 , wherein said container has a first and second positioning mechanisms, wherein said first positioning mechanism renders the container fully closed, thereby stably maintaining liquid and/or vacuum pressure inside said container and wherein said second positioning mechanism provides ventilation to the contents of said container while preserving a sterile environment inside said container. 
     
     
         147 . The kit of any one of  claims 121 - 146 , wherein said container is made of a plastic, glass, silicon, synthetic membrane, or metal. 
     
     
         148 . The kit of any one of  claims 121 - 147 , wherein said container has a treatment and/or coating of the inner surface. 
     
     
         149 . The kit of any one of  claims 121 - 148 , wherein said container is a test tube, a bottle, a well or a flask. 
     
     
         150 . The kit of any one of  claims 121 - 149 , wherein said container is vacuum sealed. 
     
     
         151 . The kit of any one of  claims 121 - 150 , wherein said container is a vacutube.